Thrombin

The cloned mMT-I/pGEX-4T-1 plasmid and mouse β₂-MG/pGEX-4T-1 plasmid were transformed into BL21(DE3)pLysS (Promega, Madison, WI, USA). The selected transformed cells were grown in 10 mL SOB medium containing 50 μg/mL ampicillin for 16 h at 37 °C until the optical density at 600 nm reached 0.3–0.4. The expression of MT and β₂-MG proteins was induced by incubation with 1 mM IPTG for 6 h at 37 °C. The cultured cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C, and the GST-fusion proteins were purified by using MagneGSTTM Protein Purification (Promega). After a dialysis against PBS, the GST-fusion MT and GST-fusion β₂-MG proteins were digested with thrombin (GE Healthcare, Buckinghamshire, UK). The lysates were loaded onto GST GraviTrapTM gravity-flow columns (GE Healthcare) to remove GST proteins, and the lysates were loaded onto a Benzamidine Sepharose 4 Fast Flow resin (GE Healthcare) to remove thrombin.
The purified recombinant MT and β₂-MG proteins were conjugated with fluorescein isothiocyanate (FITC) by Fluorescein Labeling Kit-NH2 (Dojindo, Kumamoto, Japan). For FITC-labeled albumin and Alexa-labeled transferrin, commercially available albumin-fluorescein isothiocyanate conjugate and Alexa Fluor^® 488-conjugated ChromPure Mouse Transferrin, respectively, were used.

To quantify protein yield, the cell-free reaction mixture was used to perform an EIA with anti-GII.17 mAbs (mAb 2A11 and mAb 2D11, Genecreat, China). The stool sample of positive norovirus cases were subjected to RT-PCR to obtain the ORF1 region. We then cloned capsid gene of GII.17 and expressed proteins. Mouse were immunized with GII.17 antigen and splenocytes were isolated from the mouse, then fused with myeloma. We generated a dose–response curve by using various concentrations of pure GII.17-L343/P protein binding to mAb. Pure proteins were diluted using “empty” cell-free reaction mixture to create a similar procedure to that of other cell-free proteins. Pure proteins were expressed using a pGEX-4T-1/GII.17 clade D P recombinant in E. coli strain BL21 and purified using affinity chromatography. The GST tag was removed from target proteins using Thrombin (GE Healthcare Life Sciences) at 22℃ for 20 h. Subsequently, the expression level of cell-free proteins was quantified using the “standard curve”.

NT-rfhSP-A was solubilised in 5 mM CaCl₂ and 5% glycerol (v/v), 8 M urea, pH 7.4 (solubilisation buffer) at 4 °C, overnight with mixing. NT-rfhSP-A was refolded by dialysis at 4 °C for 2 h against solubilisation buffer but with decreasing concentrations of urea (4 M, 2 M, 1 M and 0 M). After removal of precipitate, NT-rfhSP-A was purified using an IMAC purification column and cleaved through incubation with 10 units of thrombin (GE Healthcare) per mg of protein for 6 h at room temperature. rfhSP-A was purified by reapplication to an IMAC column to remove His-tagged NT. NT-rfhSP-A and rfhSP-A were analysed by SDS-PAGE under reducing conditions with subsequent Coomassie staining or analysis by Western blotting using a monoclonal mouse IgG antibody raised against nhSP-A. rfhSP-A identity and purity was confirmed by mass spectrometry using previously described methods (Simon et al., 2016 (link)).

For GST pull down assays, GST-Smad7 and 6XHis-β-catenin fusion proteins were produced in bacteria using standard protocol. Briefly, GST-Smad7- and 6XHis-β-catenin-expressing cells were sonicated and the proteins were purified with glutathione-agarose beads (Sigma-Aldrich G-4510). Protein concentrations were estimated by SDS-polyacrylamide gel electrophoresis and following Coomassie blue staining, using BSA for comparative estimation. GST-β-catenin fragments corresponding to aa (full length (FL), 1–100, 120–683, 422–683, 575–696, 575–683, 1–574) were utilized for mapping study. Five micrograms of GST FL Smad7, β-catenin (or the molar equivalent of the smaller GST, GST-β-catenin fragments), and 25 µl glutathione-agarose beads (50% slurry) were incubated in 600 µl NETN buffer (100 mM NaCl, 20 mM Tris-HCl (pH 8) 0.5 mM EDTA, 0.5% (vol/vol) NP-40) overnight at 4 °C. In experiments where GST-β-catenin was used to co-precipitate Smad7, GST-Smad7 was thrombin (GE Healthcare-27-0846-01) digested to yield the immune-complex without the GST tag (10 µl thrombin per mg fusion protein was incubated at room temperature for 17 h), and eluates were analyzed by western blotting.